引物濃度的計(jì)算:

引物保存在高濃度的狀況下比較穩(wěn)定。引物一般配制成10-50pmol/ul。一般情況下，建議將引物的濃度配制成50pmol/ul，加水的體積(微升)按下列方式計(jì)算：V (微升)= OD數(shù)*(乘)33 *(乘)*(乘)20000 / (除) 引物的分子量。引物的分子量可以從合成報(bào)告單上獲得。如果需要配制成其他濃度，按上述公式換算。

注意：1 OD260= 33 ug/ml.

引物Tm值的計(jì)算

引物設(shè)計(jì)軟件都可以給出Tm，與引物長度、堿基組成、引物使用緩沖的離子強(qiáng)度有關(guān)。

長度為25mer以下的引物，Tm計(jì)算公式為：Tm = 4℃(G + C)+ 2℃(A + T)

對于更長的寡聚核苷酸，Tm計(jì)算公式為：

Tm = 81.5 + 16.6 x Log10[Na+] + 0.41 (%GC) – 600/size

公式中，Size = 引物長度。

Tm的定義：Tm = Temperature at which 50% of a given oligonucleotide is hybridized to its complementary strand. In the absence of destabilizing agents, like formamide or urea, Tm will depend on 3 major parameters: The sequence: a GC-rich sequence has a higher melting temperature. The strand concentration: high oligonucleotide concentrations favor hybrid formation, which results in a higher melting temperature. The salt concentration: high ionic strength results in a higher Tm as cations stabilize the DNA duplexes.